Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.

Collagen fibers in the 3D tumor microenvironment (TME) exhibit complex alignment landscapes that are critical in directing cell migration through a process called contact guidance. Previous in vitro work studying this phenomenon has focused on quantifying cell responses in uniformly aligned environments. However, the TME also features short-range gradients in fiber alignment that result from cell-induced traction forces. Although the influence of graded biophysical taxis cues is well established, cell responses to physiological alignment gradients remain largely unexplored. In this work, fiber alignment gradients in biopsy samples are characterized and recreated using a new microfluidic biofabrication technique to achieve tunable sub-millimeter to millimeter scale gradients. This study represents the first successful engineering of continuous alignment gradients in soft, natural biomaterials. Migration experiments on graded alignment show that HUVECs exhibit increased directionality, persistence, and speed compared to uniform and unaligned fiber architectures. Similarly, patterned MDA-MB-231 aggregates exhibit biased migration toward increasing fiber alignment, suggesting a role for alignment gradients as a taxis cue. This user-friendly approach, requiring no specialized equipment, is anticipated to offer new insights into the biophysical cues that cells interpret as they traverse the extracellular matrix, with broad applicability in healthy and diseased tissue environments.

Expanding the applicability of multiphoton fluorescence recovery after photobleaching by incorporating shear stress in laminar flow.

Significance: Multi-photon fluorescence recovery after photobleaching (MPFRAP) is a nonlinear microscopy technique used to measure the diffusion coefficient of fluorescently tagged molecules in solution. Previous MPFRAP fitting models calculate the diffusion coefficient in systems with diffusion or diffusion in laminar flow. Aim: We propose an MPFRAP fitting model that accounts for shear stress in laminar flow, making it a more applicable technique for in vitro and in vivo studies involving diffusion. Approach: Fluorescence recovery curves are generated using high-throughput molecular dynamics simulations and then fit to all three models (diffusion, diffusion and flow, and diffusion and shear flow) to define the limits within which accurate diffusion coefficients are produced. Diffusion is simulated as a random walk with a variable horizontal bias to account for shear flow. Results: Contour maps of the accuracy of the fitted diffusion coefficient as a function of scaled velocity and scaled shear rate show the parameter space within which each model produces accurate diffusion coefficients; the shear-flow model covers a larger area than the previous models. Conclusion: The shear-flow model allows MPFRAP to be a viable optical tool for studying more biophysical systems than previous models.

Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.

In the tumor microenvironment (TME), collagen fibers facilitate tumor cell migration through the extracellular matrix. Previous studies have focused on studying the responses of cells on uniformly aligned or randomly aligned collagen fibers. However, the in vivo environment also features spatial gradients in alignment, which arise from the local reorganization of the matrix architecture due to cell-induced traction forces. Although there has been extensive research on how cells respond to graded biophysical cues, such as stiffness, porosity, and ligand density, the cellular responses to physiological fiber alignment gradients have been largely unexplored. This is due, in part, to a lack of robust experimental techniques to create controlled alignment gradients in natural materials. In this study, we image tumor biopsy samples and characterize the alignment gradients present in the TME. To replicate physiological gradients, we introduce a first-of-its-kind biofabrication technique that utilizes a microfluidic channel with constricting and expanding geometry to engineer 3D collagen hydrogels with tunable fiber alignment gradients that range from sub-millimeter to millimeter length scales. Our modular approach allows easy access to the microengineered gradient gels, and we demonstrate that HUVECs migrate in response to the fiber architecture. We provide preliminary evidence suggesting that MDA-MB-231 cell aggregates, patterned onto a specific location on the alignment gradient, exhibit preferential migration towards increasing alignment. This finding suggests that alignment gradients could serve as an additional taxis cue in the ECM. Importantly, our study represents the first successful engineering of continuous gradients of fiber alignment in soft, natural materials. We anticipate that our user-friendly platform, which needs no specialized equipment, will offer new experimental capabilities to study the impact of fiber-based contact guidance on directed cell migration.

Reference distributions of aortic calcification and association with Framingham risk score.

Evidence supporting aortic calcification as a leverageable cardiovascular risk factor is rapidly growing. Given aortic calcification's potential as a clinical correlate, we assessed granular vertebral-indexed calcification measurements of the abdominal aorta in a well curated reference population. We evaluated the relationship of aortic calcification measurements with Framingham risk scores. After exclusion, 4073 participants from the Reference Analytic Morphomic Population with varying vertebral levels were included. The percent of the aortic wall calcified was used to assess calcification burden at the L1-L4 levels. Descriptive statistics of participants, sex-specific vertebral indexed calcification measurements, relational plots, and relevant associations are reported. Mean aortic attenuation was higher in female than male participants. Overall, mean aortic calcium was higher with reference to inferior abdominal aortic measurements and demonstrated significant differences across all abdominal levels [L3 Area (mm[Formula: see text]): Females 6.34 (sd 16.60), Males 6.23 (sd 17.21); L3 Volume (mm[Formula: see text]): Females 178.90 (sd 474.19), Males 195.80 (sd 547.36); Wall Calcification (%): Females (L4) 6.97 (sd 16.03), Males (L3) 5.46 (13.80)]. Participants with elevated calcification had significantly higher Framingham risk scores compared to participants with normal calcification scores. Opportunistically measuring aortic calcification may inform further cardiovascular risk assessment and enhance cardiovascular event surveillance efforts.

Variation in aorta attenuation in contrast-enhanced CT and its implications for calcification thresholds.

BACKGROUND: CT contrast media improves vessel visualization but can also confound calcification measurements. We evaluated variance in aorta attenuation from varied contrast-enhancement scans, and quantified expected plaque detection errors when thresholding for calcification. METHODS: We measured aorta attenuation (AoHU) in central vessel regions from 10K abdominal CT scans and report AoHU relationships to contrast phase (non-contrast, arterial, venous, delayed), demographic variables (age, sex, weight), body location, and scan slice thickness. We also report expected plaque segmentation false-negative errors (plaque pixels misidentified as non-plaque pixels) and false-positive errors (vessel pixels falsely identified as plaque), comparing a uniform thresholding approach and a dynamic approach based on local mean/SD aorta attenuation. RESULTS: Females had higher AoHU than males in contrast-enhanced scans by 65/22/20 HU for arterial/venous/delayed phases (p < 0.001) but not in non-contrast scans (p > 0.05). Weight was negatively correlated with AoHU by 2.3HU/10kg but other predictors explained only small portions of intra-cohort variance (R2 < 0.1 in contrast-enhanced scans). Average AoHU differed by contrast phase, but considerable overlap was seen between distributions. Increasing uniform plaque thresholds from 130HU to 200HU/300HU/400HU produces respective false-negative plaque content losses of 35%/60%/75% from all scans with corresponding false-positive errors in arterial-phase scans of 95%/60%/15%. Dynamic segmentation at 3SD above mean AoHU reduces false-positive errors to 0.13% and false-negative errors to 8%, 25%, and 70% in delayed, venous, and arterial scans, respectively. CONCLUSION: CT contrast produces heterogeneous aortic enhancements not readily determined by demographic or scan protocol factors. Uniform CT thresholds for calcified plaques incur high rates of pixel classification errors in contrast-enhanced scans which can be minimized using dynamic thresholds based on local aorta attenuation. Care should be taken to address these errors and sex-based biases in baseline attenuation when designing automatic calcification detection algorithms intended for broad use in contrast-enhanced CTs.

High-resolution imaging of the osteogenic and angiogenic interface at the site of murine cranial bone defect repair via multiphoton microscopy.

The spatiotemporal blood vessel formation and specification at the osteogenic and angiogenic interface of murine cranial bone defect repair were examined utilizing a high-resolution multiphoton-based imaging platform in conjunction with advanced optical techniques that allow interrogation of the oxygen microenvironment and cellular energy metabolism in living animals. Our study demonstrates the dynamic changes of vessel types, that is, arterial, venous, and capillary vessel networks at the superior and dura periosteum of cranial bone defect, suggesting a differential coupling of the vessel type with osteoblast expansion and bone tissue deposition/remodeling during repair. Employing transgenic reporter mouse models that label distinct types of vessels at the site of repair, we further show that oxygen distributions in capillary vessels at the healing site are heterogeneous as well as time- and location-dependent. The endothelial cells coupling to osteoblasts prefer glycolysis and are less sensitive to microenvironmental oxygen changes than osteoblasts. In comparison, osteoblasts utilize relatively more OxPhos and potentially consume more oxygen at the site of repair. Taken together, our study highlights the dynamics and functional significance of blood vessel types at the site of defect repair, opening up opportunities for further delineating the oxygen and metabolic microenvironment at the interface of bone tissue regeneration.

Matching an immersion medium's refractive index to a cell's cytosol isolates organelle scattering.

Angularly-resolved light scattering has been proven to be an early detector of subtle changes in organelle size due to its sensitivity to scatterer size and refractive index contrast. However, for cells immersed in media with a refractive index close to 1.33, the cell itself acts as a larger scatterer and contributes its own angular signature. This whole-cell scattering, highly dependent on the cell's shape and size, is challenging to distinguish from the desired organelle scattering signal. This degrades the accuracy with which organelle size information can be extracted from the angular scattering. To mitigate this effect, we manipulate the refractive index of the immersion medium by mixing it with a water-soluble, biocompatible, high-refractive-index liquid. This approach physically reduces the amount of whole-cell scattering by minimizing the refractive index contrast between the cytosol and the modified medium. We demonstrate this technique on live cells adherent on a coverslip, using Fourier transform light scattering to compute the angular scattering from complex field images. We show that scattering from the cell: media refractive index contrast contributes significant scattering at angles up to twenty degrees and that refractive index-matching reduces such low-angle scatter by factors of up to 4.5. This result indicates the potential of refractive index-matching for improving the estimates of organelle size distributions in single cells.

Hydroxamic Acid-Modified Peptide Library Provides Insights into the Molecular Basis for the Substrate Selectivity of HDAC Corepressor Complexes.

Targeting the lysine deacetylase activity of class I histone deacetylases (HDACs) is potentially beneficial for the treatment of several diseases including human immunodeficiency virus (HIV) infection, Alzheimer's disease, and various cancers. It is therefore important to understand the function and mechanism of action of these enzymes. Class I HDACs act as catalytic components of seven large, multiprotein corepressor complexes. Different HDAC corepressor complexes have specific, nonredundant roles in the cell. It is likely that their specific functions are at least partly influenced by the substrate specificity of the complexes. To address this, we developed chemical tools to probe the specificity of HDAC complexes. We assessed a library of acetyl-lysine-containing substrate peptides and hydroxamic acid-containing inhibitor peptides against the full range of class I HDAC corepressor complexes. The results suggest that site-specific HDAC corepressor complex activity is driven in part by the recognition of the primary amino acid sequence surrounding a particular lysine position in the histone tail.

Novel Noninvasive Spinal Neuromodulation Strategy Facilitates Recovery of Stepping after Motor Complete Paraplegia.

It has been suggested that neuroplasticity-promoting neuromodulation can restore sensory-motor pathways after spinal cord injury (SCI), reactivating the dormant locomotor neuronal circuitry. We introduce a neuro-rehabilitative approach that leverages locomotor training with multi-segmental spinal cord transcutaneous electrical stimulation (scTS). We hypothesized that scTS neuromodulates spinal networks, complementing the neuroplastic effects of locomotor training, result in a functional progression toward recovery of locomotion. We conducted a case-study to test this approach on a 27-year-old male classified as AIS A with chronic SCI. The training regimen included task-driven non-weight-bearing training (1 month) followed by weight-bearing training (2 months). Training was paired with multi-level continuous and phase-dependent scTS targeting function-specific motor pools. Results suggest a convergence of cross-lesional networks, improving kinematics during voluntary non-weight-bearing locomotor-like stepping. After weight-bearing training, coordination during stepping improved, suggesting an important role of afferent feedback in further improvement of voluntary control and reorganization of the sensory-motor brain-spinal connectome.

In vitro evolution predicts emerging SARS-CoV-2 mutations with high affinity for ACE2 and cross-species binding.

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission.

Multiphoton Phosphorescence Quenching Microscopy Reveals Kinetics of Tumor Oxygenation during Antiangiogenesis and Angiotensin Signaling Inhibition.

PURPOSE: The abnormal function of tumor blood vessels causes tissue hypoxia, promoting disease progression and treatment resistance. Although tumor microenvironment normalization strategies can alleviate hypoxia globally, how local oxygen levels change is not known because of the inability to longitudinally assess vascular and interstitial oxygen in tumors with sufficient resolution. Understanding the spatial and temporal heterogeneity should help improve the outcome of various normalization strategies. EXPERIMENTAL DESIGN: We developed a multiphoton phosphorescence quenching microscopy system using a low-molecular-weight palladium porphyrin probe to measure perfused vessels, oxygen tension, and their spatial correlations in vivo in mouse skin, bone marrow, and four different tumor models. Further, we measured the temporal and spatial changes in oxygen and vessel perfusion in tumors in response to an anti-VEGFR2 antibody (DC101) and an angiotensin-receptor blocker (losartan). RESULTS: We found that vessel function was highly dependent on tumor type. Although some tumors had vessels with greater oxygen-carrying ability than those of normal skin, most tumors had inefficient vessels. Further, intervessel heterogeneity in tumors is associated with heterogeneous response to DC101 and losartan. Using both vascular and stromal normalizing agents, we show that spatial heterogeneity in oxygen levels persists, even with reductions in mean extravascular hypoxia. CONCLUSIONS: High-resolution spatial and temporal responses of tumor vessels to two agents known to improve vascular perfusion globally reveal spatially heterogeneous changes in vessel structure and function. These dynamic vascular changes should be considered in optimizing the dose and schedule of vascular and stromal normalizing strategies to improve the therapeutic outcome.

Selected 'Starter kit' energy system modelling data for selected countries in Africa, East Asia, and South America (#CCG, 2021).

Energy system modeling can be used to develop internally-consistent quantified scenarios. These provide key insights needed to mobilise finance, understand market development, infrastructure deployment and the associated role of institutions, and generally support improved policymaking. However, access to data is often a barrier to starting energy system modeling, especially in developing countries, thereby causing delays to decision making. Therefore, this article provides data that can be used to create a simple zero-order energy system model for a range of developing countries in Africa, East Asia, and South America, which can act as a starting point for further model development and scenario analysis. The data are collected entirely from publicly available and accessible sources, including the websites and databases of international organisations, journal articles, and existing modeling studies. This means that the datasets can be easily updated based on the latest available information or more detailed and accurate local data. As an example, these data were also used to calibrate a simple energy system model for Kenya using the Open Source Energy Modeling System (OSeMOSYS) and three stylized scenarios (Fossil Future, Least Cost and Net Zero by 2050) for 2020-2050. The assumptions used and the results of these scenarios are presented in the appendix as an illustrative example of what can be done with these data. This simple model can be adapted and further developed by in-country analysts and academics, providing a platform for future work.

Second-Harmonic Generation Imaging Reveals Changes in Breast Tumor Collagen Induced by Neoadjuvant Chemotherapy.

Breast cancer is the most common invasive cancer in women, with most deaths attributed to metastases. Neoadjuvant chemotherapy (NACT) may be prescribed prior to surgical removal of the tumor for subsets of breast cancer patients but can have diverse undesired and off-target effects, including the increased appearance of the 'tumor microenvironment of metastasis', image-based multicellular signatures that are prognostic of breast tumor metastasis. To assess whether NACT can induce changes in two other image-based prognostic/predictive signatures derived from tumor collagen, we quantified second-harmonic generation (SHG) directionality and fiber alignment in formalin-fixed, paraffin-embedded sections of core needle biopsies and primary tumor excisions from 22 human epidermal growth factor receptor 2-overexpressing (HER2+) and 22 triple-negative breast cancers. In both subtypes, we found that SHG directionality (i.e., the forward-to-backward scattering ratio, or F/B) is increased by NACT in the bulk of the tumor, but not the adjacent tumor-stroma interface. Overall collagen fiber alignment is increased by NACT in triple-negative but not HER2+ breast tumors. These results suggest that NACT impacts the collagenous extracellular matrix in a complex and subtype-specific manner, with some prognostic features being unchanged while others are altered in a manner suggestive of a more metastatic phenotype.

Histone H2B Deacylation Selectivity: Exploring Chromatin's Dark Matter with an Engineered Sortase.

We describe a new method to produce histone H2B by semisynthesis with an engineered sortase transpeptidase. N-Terminal tail site-specifically modified acetylated, lactylated, and ß-hydroxybutyrylated histone H2Bs were incorporated into nucleosomes and investigated as substrates of histone deacetylase (HDAC) complexes and sirtuins. A wide range of rates and site-specificities were observed by these enzyme forms suggesting distinct biological roles in regulating chromatin structure and epigenetics.

NAD(P)H autofluorescence lifetime imaging enables single cell analyses of cellular metabolism of osteoblasts in vitro and in vivo via two-photon microscopy.

Two-photon fluorescence lifetime microscopy (2P-FLIM) is a non-invasive optical technique that can obtain cellular metabolism information based on the intrinsic autofluorescence lifetimes of free and enzyme-bound NAD(P)H, which reflect the metabolic state of single cells within the native microenvironment of the living tissue. NAD(P)H 2P-FLIM was initially performed in bone marrow stromal cell (BMSC) cultures established from Col (I) 2.3GFP or OSX-mCherry mouse models, in which osteoblastic lineage cells were labelled with green or red fluorescence protein, respectively. Measurement of the mean NAD(P)H lifetime, τM, demonstrated that osteoblasts in osteogenic media had a progressively increased τM compared to cells in regular media, suggesting that osteoblasts undergoing mineralization had higher NAD+/NAD(P)H ratio and may utilize more oxidative phosphorylation (OxPhos). In vivo NAD(P)H 2P-FLIM was conducted in conjunction with two-photon phosphorescence lifetime microscopy (2P-PLIM) to evaluate cellular metabolism of GFP+ osteoblasts as well as bone tissue oxygen at different locations of the native cranial bone in Col (I) 2.3GFP mice. Our data showed that osteocytes dwelling within lacunae had higher τM than osteoblasts at the bone edge of suture and marrow space. Measurement of pO2 showed poor correlation of pO2 and τM in native bone. However, when NAD(P)H 2P-FLIM was used to examine osteoblast cellular metabolism at the leading edge of the cranial defects during repair in Col (I) 2.3GFP mouse model, a significantly lower τM was recorded, which was associated with lower pO2 at an early stage of healing, indicating an impact of hypoxia on energy metabolism during bone tissue repair. Taken together, our current study demonstrates the feasibility of using non-invasive optical NAD(P)H 2P-FLIM technique to examine cellular energy metabolism at single cell resolution in living animals. Our data further support that both glycolysis and OxPhos are being used in the osteoblasts, with more mature osteoblasts exhibiting higher ratio of NAD+/NAD(P)H, indicating a potential change of energy mode during differentiation. Further experiments utilizing animals with genetic modification of cellular metabolism could enhance our understanding of energy metabolism in various cell types in living bone microenvironment.

A correction score to compare aortic calcification in contrast enhanced and non-contrast measurements from computed tomography scans.

BACKGROUND: Aortic wall calcification shows strong promise as a cardiovascular risk factor. While useful for visual enhancement of vascular tissue, enhancement creates heterogeneity between scans with and without contrast. We evaluated the relationship between aortic calcification in routine abdominal computed tomography scans (CT) with and without contrast. METHODS: Inclusion was limited to those with abdominal CT-scans with and without contrast enhancement within 120 days. Analytic Morphomics, a semi-automated computational image processing system, was used to provide standardized, granular, anatomically indexed measurements of aortic wall calcification from abdominal CT-scans. Aortic calcification area (ACA) and aortic wall calcification percent (ACP) and were the outcomes of interest. Multiple linear regression was used to evaluate the relationship of aortic measurements. Models were further controlled for age and sex. Stratification of measurements by vertebral level was also performed. RESULTS: A positive association was observed for non-contrast calcification in ACP ß 0.74 (95% CI 0.72, 0.76) and ACA ß 0.44 (95% 0.43, 0.45). Stratified results demonstrated the highest coefficient of determination at L2 for percent and L3 for area models [R2 0.91 (ACP) 0.74 (ACA)]. Adjusted lumber-level associations between non-contrast and contrast measurements ranged from (ß 0.69-0.82) in ACP and (ß 0.37-0.54) in ACA. CONCLUSION: A straightforward correction score for comparison of abdominal aortic calcification measurements in contrast-enhanced and non-contrast scans is discussed. Correction of aortic calcification from CT scans can reduce scan heterogeneity and will be instrumental in creating larger cardiovascular cohorts as well as cardiovascular risk surveillance programs.

Spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of biomimetic nanofiber-enabled bone tissue engineering.

While extensive research has demonstrated an interdependent role of osteogenesis and angiogenesis in bone tissue engineering, little is known about how functional blood vessel networks are organized to initiate and facilitate bone tissue regeneration. Building upon the success of a biomimetic composite nanofibrous construct capable of supporting donor progenitor cell-dependent regeneration, we examined the angiogenic response and spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of cranial bone defect repair utilizing high resolution multiphoton laser scanning microscopy (MPLSM) in conjunction with intravital imaging. We demonstrate here that the regenerative vasculature can be specified as arterial and venous capillary vessels based upon endothelial surface markers of CD31 and Endomucin (EMCN), with CD31+EMCN- vessels exhibiting higher flowrate and higher oxygen tension (pO2) than CD31+EMCN+ vessels. The donor osteoblast clusters are uniquely coupled to the sprouting CD31+EMCN+ vessels connecting to CD31+EMCN- vessels. Further analyses reveal differential vascular response and vessel type distribution in healing and non-healing defects, associated with changes of gene sets that control sprouting and morphogenesis of blood vessels. Collectively, our study highlights the key role of spatiotemporal vessel type distribution in bone tissue engineering, offering new insights for devising more effective vascularization strategies for bone tissue engineering.

Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

Chronic Stress Exposure Suppresses Mammary Tumor Growth and Reduces Circulating Exosome TGF-ß Content via ß-Adrenergic Receptor Signaling in MMTV-PyMT Mice.

Preclinical models of breast cancer have established mechanistic links between psychological stress and cancer progression. However, epidemiological evidence linking stress and cancer is equivocal. We tested the impact of stress exposure in female mice expressing the mouse mammary tumor virus polyoma middle-T antigen (MMTV-PyMT), a spontaneous model of mammary adenocarcinoma that mimics metastatic hormone receptor-positive human breast cancer development. MMTV-PyMT mice were socially isolated at 6 to 7 weeks of age during premalignant hyperplasia. To increase the potency of the stressor, singly housed mice were exposed to acute restraint stress (2 hours per day for 3 consecutive days) at 8 to 9 weeks of age during early carcinoma. Exposure to this dual stressor activated both major stress pathways, the sympathetic nervous system and hypothalamic-pituitary-adrenal axis throughout malignant transformation. Stressor exposure reduced mammary tumor burden in association with increased tumor cleaved caspase-3 expression, indicative of increased cell apoptosis. Stress exposure transiently increased tumor vascular endothelial growth factor and reduced tumor interleukin-6, but no other significant alterations in immune/inflammation-associated chemokines and cytokines or changes in myeloid cell populations were detected in tumors. No stress-induced change in second-harmonic generation-emitting collagen, indicative of a switch to a metastasis-promoting tumor extracellular matrix, was detected. Systemic indicators of slowed tumor progression included reduced myeloid-derived suppressor cell (MDSC) frequency in lung and spleen, and decreased transforming growth factor ß (TGF-ß) content in circulating exosomes, nanometer-sized particles associated with tumor progression. Chronic ß-adrenergic receptor (ß-AR) blockade with nadolol abrogated stress-induced alterations in tumor burden and cleaved caspase-3 expression, lung MDSC frequency, and exosomal TGF-ß content. Despite the evidence for reduced tumor growth, metastatic lesions in the lung were not altered by stress exposure. Unexpectedly, ß-blockade in nonstressed mice increased lung metastatic lesions and splenic MDSC frequency, suggesting that in MMTV-PyMT mice, ß-AR activation also inhibits tumor progression in the absence of stress exposure. Together, these results suggest stress exposure can act through ß-AR signaling to slow primary tumor growth in MMTV-PyMT mice.

Inhibition of the mitochondrial permeability transition improves bone fracture repair.

Bone fracture is accompanied by trauma, mechanical stresses, and inflammation - conditions known to induce the mitochondrial permeability transition. This phenomenon occurs due to opening of the mitochondrial permeability transition pore (MPTP) promoted by cyclophilin D (CypD). MPTP opening leads to more inflammation, cell death and potentially to disruption of fracture repair. Here we performed a proof-of-concept study and tested a hypothesis that protecting mitochondria from MPTP opening via inhibition of CypD improves fracture repair. First, our in vitro experiments indicated pro-osteogenic and anti-inflammatory effects in osteoprogenitors upon CypD knock-out or pharmacological inhibition. Using a bone fracture model in mice, we observed that bone formation and biomechanical properties of repaired bones were significantly increased in CypD knock-out mice or wild type mice treated with a CypD inhibitor, NIM811, when compared to controls. These effects were evident in young male but not female mice, however in older (13 month-old) female mice bone formation was also increased during fracture repair. In contrast to global CypD knock-out, mesenchymal lineage-specific (Prx1-Cre driven) CypD deletion did not result in improved fracture repair. Our findings implicate MPTP in bone fracture and suggest systemic CypD inhibition as a modality to promote fracture repair.